1. Perturbed 6-Tetrahydrobiopterin Recycling via Decreased Dihydropteridine Reductase in Vitiligo: More Evidence for H2O2 Stress 
Sybille Hasse, Nicholas C.J. Gibbons, Hartmut Rokos, Lee K. Marles, Karin U. Schallreuter 
Journal of Investigative Dermatology 
Volume 122, Issue 2, Pages (February 2004)
DOI: /j X x
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
De novo synthesis/recycling/regulation of 6BH4 in the production of L-tyrosine as a substrate for melanogenesis and catecholamine synthesis. Both epidermal keratinocytes and melanocytes hold the full capacity for autocrine de novo synthesis/recycling and regulation of 6BH4 (Schallreuter et al, 1994b, c). Guanosine triphosphate (GTP) serves as a substrate for de novo synthesis where guanosine triphosphate cyclohydrolase I (GTP-CH I) is the rate-limiting step. 6BH4 acts as a cofactor for the hydroxylation of the aromatic amino acids L-phenylalanine via PAH and L-tyrosine via tyrosine hydroxylase (TH) (Kaufman, 1997). TH is the rate-limiting enzyme for catecholamine synthesis in keratinocytes (Schallreuter et al, 1992) and melanocytes.11 Gillbro JM, Marles LK, Schallreuter KU: Autocrine catecholamine synthesis and the expression of β2-adrenoceptors in human melanocytes. Pigment Cell Res 15(Suppl 9):82, 2002 (abstr.). Only recently it has been shown that TH forms L-DOPA for activation of tyrosinase in melanocytes (Marles et al, 2003). PAH produces 4α-carbinolamine as an intermediate in the recycling process of 6BH4, which is dehydrated by PCD to quinonoid dihydropterin followed by the NADH-dependent reduction via DHPR to 6BH4. In addition, 6BH4de novo synthesis is controlled by GTP-CH I feedback regulatory protein (GFRP), which upregulates GTP-CH I after binding of L-phenylalanine, whereas 6BH4 binding exerts the opposite effect. During the recycling the 7-isomer 7BH4 is nonenzymatically formed from 4α-carbinolamine. This isomer inhibits PAH as observed in untreated vitiligo (Davis et al, 1992;Schallreuter et al, 1994c;Kowlessur et al, 1996) (↑ upregulation; and ↓ downregulation in the epidermis of patients with vitiligo).
Journal of Investigative Dermatology  , DOI: ( /j X x)
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3. Figure 2
DHPR expression in human epidermal melanocytes and keratinocytes and in white blood cells (lymphocytes/monocytes). RT-PCR for DHPR mRNA in human epidermal keratinocytes (lane 3), melanocytes (lane 4), and lymphocytes/monocytes (lane 5). The primer pair amplifies a sequence of 206 bp, which is in agreement with the expected product. The product was separated in a 1.5% agarose gel (lane 1, 100-bp ladder; lane 2, negative control).
Journal of Investigative Dermatology  , DOI: ( /j X x)
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4. Figure 3
The influence of H2O2 on the Vmax of DHPR activity. Enzyme activities were determined in the presence of H2O2 in the range of 10 to 80 μm. (a) Increase of Vmax for DHPR with H2O2 concentrations of less than 30 μm. (b) Decrease of Vmax for DHPR with H2O2 concentrations of greater than 30 μm. The decrease of Vmax suggests deactivation of the enzyme. N.B., Vmax without the addition of H2O2 is 4.76 mU.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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5. Figure 4
DeepView of the short-chain helix of the active site of DHPR. (a) Native enzyme: the helix of the enzyme with the location of Lys153, Tyr149, Met146, and Met151 indicated by arrows. (b) H2O2-oxidized enzyme: a conformational change occurs owing to the oxidation of Met146 and Met151 to methionine sulfoxide affecting the orientation of Tyr149 and Lys153. This shift disrupts the integrity of the catalytic center.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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6. Figure 5
FT-Raman spectrum of native DHPR/H2O2-oxidized DHPR compared to methionine sulfoxide.Arrow, the formation of methionine sulfoxide based on the appearance of the SO stretch at 1025 cm-1, which is not detectable in native enzyme. (···) Native DHPR; (???) oxidized DHPR; (—) methionine sulfoxide standard. N.B. The standard shows in addition the presence of methionine sulfone owing to an impurity of the supplied compound. This result was also confirmed by amino acid analysis. There is no sulfone present in H2O2-oxidized DHPR.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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7. Figure 6
DHPR activities in whole-blood cells of patients with vitiligo before and after treatment with topical pseudocatalase (PC-KUS). DHPR activities (mU/mg hemoglobin) were determined in whole blood of healthy controls (n=10), untreated patients (n=27), and treated patients with vitiligo (n=10) as outlined under Materials and Methods. The result shows that DHPR activities are significantly reduced before treatment and recovered in whole blood after removal of H2O2 with pseudocatalase PC-KUS (***p<0.0001).
Journal of Investigative Dermatology  , DOI: ( /j X x)
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